Inhibition of enzymes is a major mechanism of metabolism-based drug interactions. This enzyme inhibition is typically evaluated using in vitro models during drug discovery and development, the results of which are generally submitted in regulatory submissions. One such type of enzyme inhibition is time- and cofactor (e.g., NADPH (nicotineamide adenine dinucleotide phosphate))-dependent inhibition (TDI). In this type of inhibition, the inhibitor is generated from the test compound (which may or may not be a direct inhibitor itself) during the assay by an enzyme present in the reaction mix.
Currently, the typical in vitro test for TDI is cumbersome and requires a great deal of trial and error in testing. The general test includes two separate and sequential assays: first, the IC50 shift experiment; and second, a kinetic experiment (referred to as the Kl/Kinact determination). The IC50 shift experiment typically includes an assay of various concentrations of the test compound in either the presence or absence of certain cofactors (e.g., NADPH or a NADPH regenerating system), with subcellular fractions containing the target enzyme of interest. At one predetermined time, typically about 30 minutes, a fraction of this initial incubation is diluted (typically at ratios of 1:5 to 1:20) into a secondary incubation, which contains a probe substrate (at a concentration of approximately the Km of the reaction) along with the enzyme of interest and cofactors. After a second incubation time for the diluted concentration, which is dictated by the probe substrate, the reaction is stopped and the amount of metabolite formed at each condition (each concentration, with and without the cofactor) is measured and expressed as a percent of control (control being 0% concentration of the test compound). The IC50 value, that is, the test compound concentration associated with 50% decrease in metabolite formation, may be calculated for both cofactor conditions, i.e. in the presence of the cofactor and in the absence of the cofactor. A significant difference in the IC50 values between the two cofactor conditions (IC50 value without cofactor being greater than the IC50 value with cofactor, known as an “IC50 shift”) generally indicates TDI by the test compound, and a second, follow-up experiment is conducted to determine the TDI values.
The second experiment is the Kl/Kinact determination, which measures the kinetic parameters for TDI. This assay uses the same general design as the IC50 test, assaying multiple concentrations of the test compound and an enzyme of interest in the presence of certain cofactors. In this experiment, however, at several predetermined time points, a fraction of the primary incubation is diluted into a secondary incubation, which contains a high concentration (at a concentration above the Km of the reaction, preferably 5-10× the Km) of the probe substrate, and the cofactors. After a second incubation time for the diluted concentration, the reaction is stopped and the amount of metabolite formed at each concentration is determined. For each test compound concentration, the degree of metabolism (expressed as the natural log of percent of control) is plotted against the pre-incubation time. The slope associated with each test compound concentration is determined and plotted against the test compound concentration. This data set is then used to derive kinetic parameters for TDI (Kl and Kinact) using non-linear regression or other techniques.
A problem with the above test procedure is in determining the appropriate time points at which to dilute the incubation in the Kl/Kinact assay. For example, if the test compound acts rapidly (i.e., metabolizes quickly), the dilutions should preferably take place at quicker intervals in order to achieve better slope determinations and thus, more accurate results. By contrast, if the test compound acts slower, the dilutions should desirably take place at longer intervals to permit sufficient time to achieve accurate results. The problem lies in knowing whether the test compound will act rapidly or slowly, which is not known from a typical IC50 shift experiment. Without knowing whether the test compound is rapid or slow, the kinetic experiments may require iterative trial and error in testing various sets of incubation times to get accurate results. Such trial and error is cumbersome and adds substantial cost to the procedure.